1. Field of the Invention
This invention relates to cell membrane-associated DNA binding proteins (termed DNA-R herein) from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel mammalian DNA-R gene. The invention also relates to the construction of recombinant expression constructs comprising cDNA of this novel DNA-R gene, said recombinant expression constructs being capable of expressing DNA-R protein in cultures of transformed prokaryotic and eukaryotic cells. Production of the receptor protein in such cultures is also provided, as well as the production of fragments thereof having biological activity. The invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of the novel DNA-R protein. The invention also a provides cultures of such cells producing this DNA-R protein for the characterization of novel and useful drugs. Antibodies against and epitopes of this novel DNA-R protein are also provided by the invention.
2. Background of the Invention
Extracellular DNA is a potent biological signal, being capable of initiating a wide range of immune responses in vivo and in vitro including cytokine production, influx of neutrophils, IgM secretion, B-cell proliferation and enhanced natural killer activity. These properties of extracellular DNA enable naked DNA to be used as vaccines, in some instances. In addition, extracellular DNA has been used to introduce new genetic information into cells, both in vivo and in vitro.
One important aspect of extracellular DNA transfer into mammalian cells is gene therapy. Gene transfer therapy offers the potential for treatment of a variety of diseases. The ability to provide safe, efficient, and selective in vivo gene delivery will be a critical component of future protocols. Gene transfer by injection of either plasmid DNA or DNA/liposome complexes has been demonstrated to be safe and permits expression of gene products. The uptake of DNA/liposome complexes does not depend upon specific cell-surface receptors while the mechanism mediating uptake of plasmid DNA by cells remains unknown.
In order to realize the full potential of this technology, safe delivery and efficient transgene expression of DNA in selected tissues and cells must be achieved. One approach to target DNA to tissue is the use of a receptor-mediated mechanism for the binding and internalization of DNA. Viral (retrovirus, adenovirus, adeno-associated virus) delivery of DNA to cells is via a receptor-mediated mechanism, however this technique has limited in vivo clinical application. Viral vectors have been most frequently used for ex vivo gene therapy, but the technical problems associated with transplanting transduced cells remain a serious obstacle. In addition, viral vectors have the potential to lead to virus infection or to induce an immune response against antigenic viral coat proteins.
Non-viral methods of gene delivery include liposomes, the so-called xe2x80x9cgene gunxe2x80x9d, and direct injection. Gene transfer with liposomes has been shown to result in uptake and expression of DNA. Although DNA/liposomes are effectively taken up and the cDNA on the plasmid expressed, the process is believed to be nonspecific with limited possibility of targeting selected tissue. An alternative is to administer plasmid DNA directly, without a delivery system. Cells lines in tissue culture have demonstrated in vitro uptake of plasmid DNA and the expression of the transgene on the plasmid. It has also been shown that DNA, injected directly in vivo, has been taken up and the encoded genes have been expressed. While this approach has been shown to be a safe and free from problems associated with DNA delivery by viruses, the therapeutic potential of this technology is often limited by poor transgene expression from plasmid DNA in many tissues. In addition, the mechanism by which plasmid DNA is bound and internalized into cells is not well established. Knowledge of the mechanism of plasmid DNA binding to the cell surface, and how DNA is internalized and expressed, will be critical to enhancing transgene methods that also have the potential to target selected tissues.
Antisense oligonucleotides (ODN) are another form of extracellular DNA of great importance. ODN are considered potential therapeutic agents against various pathogens and oncogenes due to their ability to specifically inhibit gene expression. When injected into tissues, ODN are internalized by cells and bind to complementary region of mRNA to inhibit translation of proteins in a highly specific manner. Different antisense ODN to HIV RNA have been shown to inhibit the infectivity of the virus in cultured human leukemia cells. Although human clinical trials using ODN to treat AIDS and other diseases are ongoing, the lack of a precise understanding of where and how gene expression is effected hinders the optimization of this technique.
Extracellular DNA is also associated with human diseases, such as cystic fibrosis. Cystic fibrosis (CF) is the most common lethal genetic disease in North America. It affects one in 2500 live births and affected individuals have a median life expectancy of 28 years (Davis et al., 1996, Amer. J. Respir. Crit Care Med. 157: 1234-1239). There is a growing body of evidence showing that inflammation, particularly the injurious products of neutrophils, may be responsible for lung damage (Doring, 1997, Ped. Pulmonol. Supp. 16: 271-272); it is now recognized that most of the morbidity and over 90% of the mortality results from chronic progressive inflammation of the lungs. Corticosteroids have abroad anti-inflammatory effect, particularly on neutrophils. A multicenter trial showed beneficial effects of oral corticosteroids on lung function. However, adverse effects such as growth retardation, glucose abnormalities and cataracts prelude this treatment as a long-term option (Eigen et al., 1995, J. Ped. 126: 515-523). The nonsteroidal anti-inflammatory drug, ibuprofen, has also been studied (Konstan et al., 1995, N. Engl. J. Med. 332: 848-854). The drug is beneficial, but continued monitoring is needed to determine the safety of long-term, high dose therapy. Other therapies that treat the injurious products of neutrophils, for example, antiproteases and antioxidants, are currently under investigation (Konstan, 1998, Clin. Chest Med. 19: 505-513).
The vicious airway fluid characteristic of CF can obstruct airflow and provides a viable growth medium for pathogenic bacteria, and cell lysis of these bacteria can produce extracellular DNA that causes inflammation. Recombinant human Dnase (rhDNase) has been clinical use since 1994 (Kontsan, 1998, ibid.). The rhDNase, administered by inhalation, has been used to cleave the extracelular airway DNA and reduce the viscosity of the airway fluid. Treatment with rhDNase produces a small improvement in lung function (Cramer and Bosso, 1996, Ann. Pharmacol. 30: 656-661). However, when treatment is stopped, patients can deteriorate to a point below their previous baseline (Bush, 1998, Ped. Pulmonol. 25: 79-82). In addition, a recent report showed that despite improvements in lung function, there were no changes in airway inflammation (Henry et al., 1998, Ped. Pulmonol. 26: 97-100). Although the DNA is broken down by the Dnase, it is not entirely degraded, and hydrolized fragments are still potentially immunostimulatory and can contribute to inflammation. Thus rhDNase may be masking the process of on-going lung destruction.
There are also a variety of conventional treatments for CF including physiotherapy, nutritional support and drugs (Bilton and Mahadeva, 1997, J. Royal Soc. Med. 90: Suppl.31,2-5). Because the events that trigger and sustain inflammation in patients with CF are not clearly understood, a variety of approaches have been developed to treat different components of the disease. Antibiotics, anti-inflammatories, and therapies to reduce the viscosity of the airway fluid are all approaches that are being used and investigated. Aggressive antibiotic therapy has helped the acute control of infection, but rarely if ever are the bacteria in the airways of patients with CF completely eradicated. These pathogenic bacteria chronically stimulate and exacerbate inflammation. Although some of the currently-available treatments can help to alleviate symptoms and slow the progression of disease, none of the current treatments can prevent ultimate respiratory failure.
One important clinical observation is that greatly increased amounts of extracellular DNA, of host and bacterial origin, are present in the airway of patients with cystic fibrosis. Recent investigation has demonstrated that extracellular DNA, purified from sputum of patients with CF, will directly induce inflammation in the mouse lung (Schwartz et al., 1997, J. Clin. Invest. 100: 68-73). The DNA purified from the sputum of patients with cystic fibrosis has been shown to be composed primarily of host-derived DNA and only a small fraction appears to be bacterial DNA (Schwartz et al., 1997, ibid.). One possible explanation is that extracellular DNA binds to immune lung cells in the lungs and induces the secretion of pro-inflammatory cytokines and neutrophic migration to the lung, leading to severe airway inflammation. Extracellular DNA binding to immune cells in the lung, such as alveolar macrophages are stimulated to produce pro-inflammatory cytokines that recruit and activate neutrophils leading to inflammation. When these neutrophils undergo apoptosis and release their DNA the cycle is repeated and inflammation is maintained or increased. Thus, methods and reagents that block DNA binding to cytokine producing cells may therefore provide better treatment of CF patients than are currently available.
Although there have been several reports in the art that DNA could bind to cell surfaces (Bennett, 1993, Antisense Res. Develop. 3: 235-241; Bennett et al., 1986, J. Rheumatol. 13: 679-685; Gabor and Bennett, 1984, Biochem Biophys. Res. Commun. 122:1034-1039; Hefeneider et al., 1990, J. Invest. Dermatol. 94: 79S-84S; Bennett et al., 1987, J. Exp. Med. 166: 850-863; Bennett et al., 1991, Clin. Exp. Immunol. 86: 374-379; Bennett et al., 1992, Clin. Exp. Immunol 90: 428-433; Bennett et al., 1985, J. Clin. Invest. 76: 2182-2190; Hefeneider et al., 1992, Lupus 1: 167-173; Hefeneider et al., 1992, Clin. Immunol.Immmunopath. 63: 245-251; Reid and Chalson, 1979, Intl. Rev. Cytol. 60: 27-52; Lerner et al., 1971, Proc. Natl. Acad. Sci. USA 68: 1212-1216; Pancer et al., 1981, J. Immunol. 127: 98-104; Meinke and Goldstein, 1974, J. Molec. Biol. 86: 757-773; Sudar et al, 1986, Cell. Molec. Biol. 32: 87-91; Gasparro et al, 1990, Photochem and Photobiol. 52: 315-321; Emlen et al., 1988, Amer. J. Pathol. 133: 54-60), the art lacks an understanding of how cells mediate extracellular DNA binding. Thus, an understanding of the mechanisms by which eukaryotic cells, particularly mammalian cells, take up extracellular DNA would be important in improving a variety of biological processes.
The present invention relates to the cloning, expression and functional characterization of a mammalian DNA-R gene. The invention comprises nucleic acids having a nucleotide sequence of a novel mammalian DNA-R gene. The nucleic acids provided by the invention comprise a complementary DNA (cDNA) copy of the corresponding mRNA transcribed in vivo from the DNA-R genes of the invention. In a preferred embodiment, the mammalian DNA-R is a human DNA-R. Also provided are the deduced amino acid sequence of the cognate proteins of the cDNAs provided by the invention, methods of making said cognate proteins by expressing the cDNAs in cells transformed with recombinant expression constructs comprising said cDNAs, and said recombinant expression constructs and cells transformed thereby.
This invention in a first aspect provides nucleic acids, nucleic acid hybridization probes, recombinant eukaryotic expression constructs capable of expressing the DNA-Rs of the invention in cultures of transformed cells, and such cultures of transformed eukaryotic cells that synthesize the DNA-Rs of the invention. In another aspect, the invention provides homogeneous compositions of the DNA-R proteins of the invention, homogeneous compositions of fragments of said DNA-R, most preferably a fragment comprising amino acids 1-575 of the DNA-R, as well as fusion proteins between the DNA-R or fragments thereof and, inter alia, epitope markers, and membrane preparations from cells expressing the DNA-R proteins of the invention, and also antibodies against and epitopes of the DNA-R proteins or fragments thereof of the invention. The invention in another aspect provides methods for making said homogenous preparations and membrane preparations using cells transformed with the recombinant expression constructs of the invention and expressing said DNA-R proteins thereby. Methods for characterizing the receptor and biochemical properties of these receptor proteins and methods for using these proteins in the development of agents having pharmacological uses related to the DNA-R of the invention are also provided.
In a first aspect, the invention provides a nucleic acid having a nucleotide sequence encoding a mammalian DNA-R. In a first preferred embodiment, the nucleic acid encodes a human DNA-R. In this embodiment of the invention, the nucleotide sequence comprises 4351 nucleotides of human DNA-R cDNA comprising 3576 nucleotides of coding sequence, 601 nucleotides of 5xe2x80x2 untranslated sequence and 177 nucleotides of 3xe2x80x2 untranslated sequence. In this embodiment of the invention, the nucleotide sequence of the DNA-R is the nucleotide sequence depicted in FIG. 1 (SEQ ID No:1). The sequence shown in FIG. 1 will be understood to represent one specific embodiment of a multiplicity of nucleotide sequences that encode the human DNA-R amino acid sequence of 1192 amino acids (SEQ ID No.:2) of the invention and that these different nucleotide sequences are functionally equivalent and are intended to be encompassed by the claimed invention. In addition, it will be understood that different organisms and cells derived therefrom express preferentially certain transfer RNAs (tRNAs) corresponding to subsets of the degenerate collection of tRNAs capable of encoding certain of the naturally-occurring amino acids, and that embodiments of the multiplicity of nucleotide sequences encoding the amino acid sequence of the human DNA-R protein of the invention that are optimized for expression in specific prokaryotic and eukaryotic cells are also encompassed by the claimed invention. Isolated nucleic acid derived from human genomic DNA and isolated by conventional methods using the human cDNA provided by the invention is also within the scope of the claimed invention. Finally, it will be understood that allelic variations of the human DNA-R, including naturally occurring and in vitro modifications thereof are within the scope of this invention. Each such variant will be understood to have essentially the same amino acid sequence as the sequence of the human DNA-R disclosed herein.
Mammalian DNA-R proteins corresponding to the human cDNA of the invention are a second aspect of the claimed invention. In a first embodiment, the mammalian DNA-R protein is a human DNA-R having a deduced amino acid sequence shown in FIG. 1 (SEQ ID No.:2). In a second embodiment is provided said human DNA-R protein comprising a membrane preparation from a cell, most preferably a recombinant cell, expressing a nucleic acid encoding a human DNA-R of the invention.
As provided in this aspect of the invention is a homogeneous composition of a mammalian DNA-R having a molecular weight of about 150 kD or derivative thereof that is a human DNA-R having an amino acid sequence shown in FIG. 1 and identified by SEQ ID No.:2, said size being understood to be the predicted size of the protein before any post-translational modifications thereof. Also provided is a homogeneous composition of An amino-terminal fragment of the human DNA-R comprising amino acid residues 1-575 of the sequence identified as SEQ ID No.:2. Species of the protein genetically engineered to lack the transmembrane region of the DNA-R as described herein, and thereby providing soluble forms of the DNA-R of the invention, are also within the scope of this aspect of the invention and are provided herein.
This invention provides both nucleotide and amino acid probes derived from the sequences herein provided. The invention includes probes isolated from either cDNA or genomic DNA, as well as probes made synthetically with the sequence information derived therefrom. The invention specifically includes but is not limited to oligonucleotide, nick-translated, random primed, or in vitro amplified probes made using cDNA or genomic clone of the invention encoding a mammalian DNA-R or fragment thereof, and oligonucleotide and other synthetic probes synthesized chemically using the nucleotide sequence information of cDNA or genomic clone embodiments of the invention.
It is a further object of this invention to provide such nucleic acid hybridization probes to determine the pattern, amount and extent of expression of the DNA-R gene in various tissues of mammals, including humans. It is also an object of the present invention to provide nucleic acid hybridization probes derived from the sequences of mammalian DNA-R genes of the invention to be used for the detection and diagnosis of genetic diseases. It is an object of this invention to provide nucleic acid hybridization probes derived from the nucleic acid sequences of the mammalian DNA-R genes herein disclosed to be used for the detection of novel related receptor genes.
The present invention also includes synthetic peptides made using the nucleotide sequence information comprising the cDNA embodiments of the invention. The invention includes either naturally occurring or synthetic peptides which may be used as antigens for the production of DNA-R-specific antibodies, or useful as competitors of DNA-R molecules for nucleic acid binding, or to be used for the production of inhibitors of nucleic acid binding to such DNA-R molecules.
The present invention also provides antibodies against and epitopes of the mammalian DNA-R molecules of the invention. It is an object of the present invention to provide antibodies that are immunologically reactive to the DNA-Rs of the invention. It is a particular object to provide monoclonal antibodies against these DNA-Rs. Hybridoma cell lines producing such antibodies are also objects of the invention. It is envisioned at such hybridoma cell lines may be produced as the result of fusion between a non-immunoglobulin producing mouse myeloma cell line and spleen cells derived from a mouse immunized with a cell line which expresses antigens or epitopes of a mammalian DNA-R of the invention. The present invention also provides hybridoma cell lines that produce such antibodies, and can be injected into a living mouse to provide an ascites fluid from the mouse that is comprised of such antibodies. It is a further object of the invention to provide immunologically-active epitopes of the mammalian DNA-R proteins of the invention. Chimeric antibodies immunologically reactive against the DNA-R proteins of the invention are also within the scope of this invention.
The present invention provides recombinant expression constructs comprising a nucleic acid encoding a mammalian DNA-R of the invention wherein the construct is capable of expressing the encoded DNA-R in cultures of cells transformed with the construct. A preferred embodiment of such constructs comprises a human DNA-R cDNA depicted in FIG. 1 (SEQ ID No.:1), such constructs being capable of expressing the human DNA-R encoded therein in cells transformed with the construct. Also provided are recombinant expression constructs encoding fragments of said DNA-R, most preferably an amino-terminal fragment comprising amino acid residues 1-575 and fragments genetically engineered to lack the transmembrane domain of said DNA-R, there by providing for production of soluble forms of the DNA-R. In alternative embodiments, the recombinant expression construct encodes a DNA-R fused to epitope sequences recognized by conventional antibodies known in the art. In each instance, the recombinant expression constructs of the invention are capable of expressing the human DNA-R encoded therein or fragment thereof in cells transformed with the construct.
The invention also provides prokaryotic and more preferably eukaryotic cells transformed with the recombinant expression constructs of the invention, each such cells being capable of and indeed expressing the mammalian DNA-R or fragment or epitope-modified species encoded in the transforming construct, as well as methods for preparing mammalian DNA-R proteins using said transformed cells.
The present invention also includes within its scope protein preparations of prokaryotic and eukaryotic cell membranes containing the DNA-R protein of the invention, or fragment or epitope-modified species thereof, derived from cultures of prokaryotic or eukaryotic cells, respectively, transformed with the recombinant expression constructs of the invention.
The invention also provides methods for screening compounds for their ability to inhibit, facilitate or modulate the biochemical activity of the mammalian DNA-R molecules of the invention, in particular nucleic acid binding thereto. In preferred embodiments, the methods of the invention relate to binding of DNA, particularly double-stranded DNA, and oligonucleotides. The methods of the invention are particularly directed towards identifying compounds that influence DNA or oligonucleotide uptake into cells expressing the DNA-R. In preferred embodiments, the compounds identified by the methods of the invention influence DNA or oligonucleotide uptake by pinocytosis or endocytosis. In preferred embodiments, the compounds influence DNA or oligonucleotide uptake by increasing the amount of DNA or oligonucleotide that reaches the nucleus of the cell in a form that can be expressed therein. Preferred compounds of the invention are identified by detecting increased uptake or increased expression of a gene, most preferably a reporter gene, encoded by said DNA. In preferred embodiments, cells transformed with a recombinant expression construct of the invention are contacted with such a compound, and the amount of DNA or oligonucleotide taken up by the cell, or the frequency or amount of gene expression, most preferably reporter gene expression, in the cell is assayed.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.